In STED, GSD and RESOLFT scanning fluorescence light microscopy it is known to form a three-dimensional light intensity distribution comprising a spatially limited area of minimum light intensity that is enclosed by areas of higher light intensity of fluorescence inhibiting light by means of shaping the wave fronts of a beam of coherent fluorescence inhibiting light by means of a phase plate. One known phase plate for this application is a so-called phase clock which provides a phase shift increasing from zero to 2π over one turn about the optical axis, corresponding to an optical path difference of λ which is the wavelength of the fluorescence inhibiting light. When the beam of the fluorescence inhibiting light, after passing this phase clock and after the fluorescence inhibiting light being circularly polarized, is focused into a focal area by means of an objective, the resulting light intensity distribution comprises a zero point in the focal area enclosed by a torus of higher light intensity around the optical axis, which is often called a doughnut. By means of this light intensity distribution of fluorescence inhibiting light the spatial resolution of scanning fluorescence light microscopy will be enhanced within the focal plane of the objective, i.e. in x- and y-directions, but not normal to the focal plane, i.e. in z-direction.
In so-called 4Pi STED scanning fluorescence light microscopy, it is known to use an apparatus for forming a three-dimensional light intensity distribution comprising a spatially limited area of minimum light intensity that is enclosed by areas of higher light intensity of the fluorescence inhibiting light, which comprises two objectives facing each other on a common optical axis. These objectives focus light coming out of opposite directions into a common focal area. Fluorescence inhibiting light from a coherent light source is split into a pair of coherent light beams and each of the two objectives focuses one of these coherent light beams into the common focal area. If the beam paths of the two coherent light beams which each extend from the beam splitters through one of the objectives and to the common focal area differ in optical length by λ(2n+1)/2, the light intensities of the coherent light beams extinguish each other in a partial area of the focal area extending along the focal planes of the objectives. This area of minimum light intensity is enclosed by areas of higher light intensity of the fluorescence inhibiting light on both sides in direction of the common optical axis. Thus, in 4Pi STED fluorescence light microscopy the spatial resolution is enhanced in the z-direction of the common optical axis but neither in x- nor in y-direction. This spatial resolution in x- and y-direction may be enhanced by means of a confocal detection arrangement of the fluorescence light registered from the respective sample.
Dyba M et al “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment”, NEW JOURNAL OF PHYSICS, INSTITUTE OF PHYSICS PUBLISHING, BRISTOL, GB, besides standard 4Pi STED scanning fluorescence light microscopy, report a study of phase modifications of the wavefront of the stimulating beam that strengthen weakly transferred frequencies within the optical transfer-function of a 4Pi STED microscope. The enlarged bandwidth shall enable the separation of objects at 76 nm axial distance.
Hongki Yoo, Incheon Song, Taehoon Kim, Daegab Gweon “Effects of a pupil filter on stimulated emission depletion microscopy”, THREE-DIMENSIONAL AND MULTIDIMENSIONAL MICROSCOPY, IMAGE ACQUISITION AND PROCESSING XIII, BELLINGHAM, USA, report effects of a pupil filter on the performances of STED-scanning fluorescence light microscopy. Using a half-coated phase plate, a zero-centered spot is made with a narrow and steep gap at the center to achieve a high lateral resolution. Numerical and experimental results show that by simply inserting a central obstacle as a pupil filter, it is possible to reduce the central gap of the zero-centered spot. However, in order to compensate inevitable loss of light, which is blocked by the obstacle, increased laser power is required.
Haeberle O et al “Improving the lateral resolution in confocal fluorescence microscopy using laterally interfering excitation beams”, OPTICS COMMUNICATIONS, NORTH-HOLLAND PUBLISHING CO., AMSTERDAM, NL, disclose an STED-scanning fluorescence light microscopy variant in which the lateral resolution is based on laterally interfering beams. A half phase plate is used to modify the illumination, combined with a laterally offset detection. Another approach uses several excitation beams, slightly shifted and properly dephased, to decrease the lateral extension of the point spread function (PSF).
WO 2011/086519 A1 discloses an STED microscopy system in which an optical element is supplied for focusing a first excitation and a second depletion beam on an object, thereby defining a common optical path for both the first and the second beam. A phase modifying member is inserted in the common optical path, and the phase modifying member is optically configured for leaving the wavefront of the first beam substantially unchanged, and for changing the wavefront of the second beam so as to create an undepleted region of interest in the object.
In so-called isoSTED scanning fluorescence light microscopy which is, for example, described in DE 10 2008 019 957 A1, an apparatus including a phase plate and an apparatus including two objectives facing each other are combined for forming a three-dimensional light intensity distribution of fluorescence inhibiting light enhancing the spatial resolution by means of the fluorescence inhibiting light both in x- and y- and in z-directions. In this combined apparatus, it has to be cared for that the light of the beam of coherent light passing through the phase plate and then being focused into the focal area does not interfere with the light of the pair of coherent light beams focused and superimposed by the two objectives. If this care is not taken, the light intensities of the two individual three-dimensional light intensity distributions will not simply add up but may interfere with each other, i.e. mutually affect each other.
An apparatus which would allow for generating a three-dimensional light intensity distribution comprising an even steeper light intensity gradient between the area of minimum light intensity to the enclosing areas of higher light intensity than in isoSTED considering same light intensities would be desirable.